Chronic pain is a significant health care problem, adversely affecting the quality of life of millions of people worldwide and costing billions of dollars each year in lost productivity. Research conducted in the past decade has revealed that pain facilitatory neurons in the brainstem play an important role in the development and particularly the maintenance of chronic pain after peripheral injury. However, the exact mechanisms remain elusive. Recent behavioral studies by this and other laboratories indicate that release of substance P in the rostral ventromedial medulla (RVM) mediates the thermal and mechanical hypersensitivity that follow inflammatory injury of the hindpaw. Thus, local antagonism of neurokinin-1 receptors in the RVM was sufficient to reversal hyperalgesia and hypersensitivity induced by complete Freund's adjuvant (CFA). Whole cell patch clamp recordings further revealed that the facilitation of glutamatergic inputs to spinally projecting RVM neurons observed in CFA-treated rats was dependent on release of substance P. Finally, pharmacological antagonism of postsynaptic N-methyl-D-aspartate receptors (NMDAR) was without effect, whereas extracellular application of a NMDAR antagonist prevented the facilitation. These data lead to the hypothesis that NMDARs situated presynaptically on the terminals of substance P afferents to RVM neurons function to promote the release of substance P, and that the up-regulation of these receptors after inflammatory injury sets in motion a positive feed-forward mechanism to sustain activity in pain facilitatory neurons in the RVM. Three complementary sets of studies are proposed to begin to test this hypothesis. First, the ability of glutamate or NMDA to promote the release of substance P will be investigated in isolated presynaptic terminals (synaptosomes) from the RVM of rats that received an intraplantar injection of saline- or CFA four hrs, four days or two weeks earlier. The subtype of NMDA receptor will be determined using subunit specific antagonists. Second, inflammation-induced changes in NMDA receptor subunit transcription, translation, surface expression and phosphorylation state will be assessed in synaptosomes of the RVM using quantitative real time PCR and western blotting with subunit specific antibodies, phospho-specific antibodies and biotinylation probes. Lastly, immunohistochemical methods will be used to confirm changes in the expression of NMDAR subunits in substance P-immunoreactive presynaptic structures in the RVM after CFA-treatment. Results obtained from these studies will further our understanding of the mechanism by which supraspinal nuclei function to maintain chronic pain after injury, as well as probe the activity-dependent plasticity of presynaptic NMDAR. This information could aid the development of targeted, centrally active therapeutics for the treatment of persistent pain states. The approaches selected to test this hypothesis will provide the applicant with broad-based training in neurochemical, pharmacological, molecular and immunohistochemical approaches in preparation for a career as an independent investigator. PUBLIC HEALTH RELEVANCE: This project seeks to further elucidate the mechanisms by which brainstem neurons function to initiate and then maintain chronic pain after peripheral inflammatory injury. As chronic pain affects millions of people in the USA alone, it is in the public's interest to find better ways to treat this condition. Results from this proposal could potentially aid in understanding and developing novel, centrally acting therapeutics for the treatment of chronic pain.